ABSTRACT
OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (P=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.
Subject(s)
Aortic Dissection/genetics , Arteries/pathology , Collagen Type V/genetics , Ehlers-Danlos Syndrome/genetics , Fibromuscular Dysplasia/genetics , Polymorphism, Single Nucleotide , Adult , Aortic Dissection/diagnostic imaging , Aortic Dissection/pathology , Arteries/diagnostic imaging , Ehlers-Danlos Syndrome/diagnostic imaging , Ehlers-Danlos Syndrome/pathology , Female , Fibromuscular Dysplasia/diagnostic imaging , Fibromuscular Dysplasia/pathology , Genetic Predisposition to Disease , Haplotypes , Humans , Male , Middle Aged , Phenotype , Young AdultABSTRACT
Obesity-induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c+ adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs) and Toll-like receptor 4 (TLR4), and the downstream TIR domain-containing adapter protein-inducing interferon-ß (TRIF)- and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis is unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing pro-inflammatory CD11c+ myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4-/- and WT mice indicated that TLR4 is required for HFD-induced myelopoiesis and production of CD11c+ ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte macrophage progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4-/-, Myd88-/-, and Trif-/- mice on HFD demonstrated that TLR4 plays a role in the production of CD11c+ ATMs, and both Myd88-/- and Trif-/- mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c+ ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88- and TRIF-mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization.
Subject(s)
Adaptor Proteins, Vesicular Transport/immunology , CD11c Antigen/immunology , Macrophages/pathology , Myeloid Differentiation Factor 88/immunology , Myelopoiesis , Obesity/pathology , Toll-Like Receptor 4/immunology , Adipose Tissue/immunology , Adipose Tissue/pathology , Animals , Diet, High-Fat/adverse effects , Inflammation/etiology , Inflammation/immunology , Inflammation/pathology , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Obese , Obesity/complications , Obesity/etiology , Obesity/immunologyABSTRACT
Transforming growth factor-beta3 (TGF-ß3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-ß3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-ß activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-ß signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-ß signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-ß signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.
Subject(s)
MAP Kinase Kinase Kinases/metabolism , Palate/embryology , Palate/metabolism , Signal Transduction , Smad4 Protein/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta3/metabolism , Animals , Apoptosis/genetics , Cell Fusion , Cell Proliferation , Crosses, Genetic , Embryo, Mammalian/metabolism , Enzyme Activation , Epithelial Cells/metabolism , Epithelium/metabolism , Female , Gene Deletion , Gene Expression Regulation, Developmental , Male , Matrix Metalloproteinase 13/metabolism , Mice, Knockout , Models, Biological , Mutation/genetics , Organ Specificity , Palate/abnormalities , Palate/enzymologyABSTRACT
BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease.
Subject(s)
Activin Receptors, Type I/metabolism , Arteries/embryology , Body Patterning/physiology , Bone Morphogenetic Proteins/metabolism , Gene Expression Regulation, Developmental/genetics , Heart/embryology , Morphogenesis/physiology , Activin Receptors, Type I/genetics , Animals , Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Protein Receptors, Type I/metabolism , Cell Differentiation/physiology , Gene Knock-In Techniques , Genetic Vectors/genetics , Immunohistochemistry , In Situ Hybridization , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Morphogenesis/genetics , Myocardium/cytology , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
BACKGROUND: The role of Smad-independent TGF-ß signaling in craniofacial development is poorly elucidated. RESULTS: In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-ß signaling. CONCLUSION: Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. SIGNIFICANCE: Understanding the mechanisms of TGF-ß signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-ß superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called "canonical") signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-ß-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-ß superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-ß- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFßRI and Tak1 kinases mediate both overlapping and distinct TGF-ß2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-ß superfamily signaling required for normal craniofacial development.
Subject(s)
MAP Kinase Kinase Kinases/physiology , Neural Crest/cytology , Protein Processing, Post-Translational , Smad Proteins/metabolism , Amino Acid Motifs , Animals , Cells, Cultured , Cleft Palate/enzymology , Cleft Palate/genetics , Ectoderm/cytology , Female , Gene Expression Regulation, Developmental , Head/embryology , MAP Kinase Kinase Kinases/deficiency , MAP Kinase Kinase Kinases/genetics , Male , Mandible/abnormalities , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Smad Proteins, Receptor-Regulated/metabolism , TGF-beta Superfamily Proteins/physiology , Wnt1 Protein/genetics , Wnt1 Protein/metabolismABSTRACT
UNLABELLED: Palatogenesis, the formation of the palate, is a dynamic process regulated by a complex series of context-dependent morphogenetic signaling events. Many genes involved in palatogenesis have been discovered through the use of genetically manipulated mouse models as well as from human genetic studies, but the roles of these genes and their products in signaling networks regulating palatogenesis are still poorly known. In this review, we give a brief overview on palatogenesis and introduce key signaling cascades leading to formation of the intact palate. Moreover, we review conceptual differences between pathway biology and network biology and discuss how some of the recent technological advances in conjunction with mouse genetic models have contributed to our understanding of signaling networks regulating palate growth and fusion. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.
Subject(s)
Palate/metabolism , Animals , Embryonic Development , Epithelial Cells/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Palate/growth & development , Signal TransductionABSTRACT
Tgfb3 is strongly and specifically expressed in the epithelial tips of pre-fusion palatal shelves where it plays a critical non-redundant role in palatal fusion in both medial edge epithelial (MEE) cells and in a thin layer of flattened peridermal cells that covers the MEE. It is not known how Tgfb3 expression is regulated in these specific cell types. Using comparative genomics and transgenic reporter assays, we have identified cis-regulatory elements that could control Tgfb3 expression during palatogenesis. Our results show that a 61-kb genomic fragment encompassing the Tgfb3 gene drives remarkably specific reporter expression in the MEE and adjacent periderm. Within this fragment, we identified two small, non-coding, evolutionarily conserved regions in intron 2 of the neighboring Ift43 gene, and a larger region in the intervening sequence between the Ift43 and Tgfb3 genes, each of which could target reporter activity to the tips of pre-fusion/fusing palatal shelves. Identification of the cis-regulatory sequences controlling spatio-temporal Tgfb3 expression in palatal shelves is a key step toward understanding upstream regulation of Tgfb3 expression during palatogenesis and should enable the development of improved tools to investigate palatal epithelial fusion.